Scientists and clinicians face a number of challenges in their attempts to develop active agents into forms suited for delivery to a patient. Active agents that are polypeptides, for example, are often delivered by injection rather than orally. In this way, the polypeptide is introduced into the systemic circulation without exposure to the proteolytic environment of the stomach. Injection of polypeptides, however, has several drawbacks. For example, many polypeptides have a relatively short half-life, thereby necessitating the need for repeated injections, which are often inconvenient and painful. Moreover, some polypeptides may elicit one or more immune responses with the consequence that the patient's immune system may be activated to degrade or inactivate the polypeptide. Thus, the delivery of polypeptides and other active agents is often problematic even when these agents are administered by injection.
Some success in addressing the problems of delivering active agents via injection has been achieved. For example, conjugating the active agent to a water-soluble polymer has resulted in polymer-active agent conjugates having reduced immunogenicity and antigenicity. In addition, these conjugates often have greatly increased half-lives compared to their unconjugated counterparts as a result of decreased clearance through the kidney and/or decreased enzymatic degradation in vivo. As a result of having a greater half-life, the conjugate requires less frequent dosing, which in turn reduces the overall number of painful injections and inconvenient visits to a health care professional. Moreover, active agents that are only marginally soluble often demonstrate a significant increase in water solubility when conjugated to a water-soluble polymer.
Due to its documented safety as well as its approval by the FDA for both topical and internal use, poly(ethylene glycol) has been conjugated to active agents. When an active agent is conjugated to a polymer of poly(ethylene glycol) or “PEG”, the conjugated active agent is conventionally referred to as having been “PEGylated.” The commercial success of PEGylated active agents attests to their value. Exemplary commercially available PEGylated polypeptides include PEGASYS® PEGylated interferon alpha-2a (Hoffmann-La Roche, Nutley, N.J.), PEG-INTRON® PEGylated interferon alpha-2b (Schering Corp., Kennilworth, N.J.), SOMAVERT® PEGylated human growth hormone receptor antagonist, and NEULASTA® PEG-filgrastim (Amgen Inc., Thousand Oaks, Calif.). PEGylated small molecules such as distearoylphosphatidylethanolamine (Zalipsky (1993) Bioconjug. Chem. 4(4):296–299) and fluorouracil (Ouchi et al. (1992) Drug Des. Discov. 9(1):93–105) have also been prepared.
Despite these successes, conjugation of a polymer to an active agent remains challenging. In particular, the conjugation reaction is relatively imprecise in that a relatively disperse mixture of products results. For example, the conjugation reaction often results in a mixture of singly substituted, disubstituted, and polysubstituted conjugate forms. Moreover, the possible number of different conjugate forms increases inasmuch as different attachment sites of an active agent can result in different arrangements of attachments. For example, a singly substituted active agent having three different sites for attachment to a polymer can have three different forms: each form having the single polymer attached to one of the three different attachment sites. Recognizing that each conjugate form can have a unique pharmacodynamic and pharmacokinetic profile only serves to underscore the complexity associated with providing polymer-active agent conjugates that are suited for use as therapeutic agents. Thus, there is a desire to provide polymeric reagents that are “site-directed” so that the desired conjugates can be more predictably formed.
Some degree of selectivity in the conjugation reaction is achieved based on the presence of the particular functional group present on the polymeric reagent. For example, poly(ethylene glycol) derivatives bearing an aldehyde derivative (shown below) undergo reductive amination reactions with primary amines of, for example, a polypeptide.
                a poly(ethylene glycol) derivative bearing an aldehyde derivative        wherein (n) represents the number of repeating ethylene oxide units        
As a consequence, a degree of selectivity is achieved in that conjugation typically occurs only between the relatively reactive and readily accessible primary amine groups of the polypeptide and the aldehyde of the polymeric reagent. For polypeptides having many relatively reactive primary amine groups (e.g., a polypeptide having many primary amine-containing lysine residues), however, the conjugation reaction nonetheless results in a relatively polydisperse mixture of conjugate forms.
In order to provide an alternative to polymeric reagents bearing aldehyde derivatives, the present invention provides polymeric reagents comprising a ketone or a related functional group (e.g., a ketone hydrate, thione, monothiohydrate, dithiohydrate, hemiketal, monothiohemiketal, dithiohemiketal, ketal, or dithioketal).
Others have described polymers comprising a ketone or a related functional group for use in variety of contexts. Typically, however, the described polymers are unsuited for conjugation.
For example, WO 96/33156 describes poly(alkylene glycol) derivatives of benzophenone and related aromatic moieties. The derivatives are described as being useful as photoinitiators. Aromatic rings, however, are preferably absent in polymeric reagents useful for conjugation to an active agent because the hydrophobic nature of aromatic moieties undesirably decreases aqueous solubility. In addition, aromatic rings contained in a conjugate may be metabolized in vivo into arene oxide intermediates, which can covalently bind with nucleophilic groups present on proteins, DNA and RNA, thereby leading to cellular toxicity. See Daly et al. (1979) Experientia 28(10):1129–1149.
U.S. Pat. No. 5,149,806 describes a carbon acid useful as a Michael donor. One of the described structures is provided below:
Even if structures like these could be used for conjugation reactions, the propylene oxide polymers are not suitably water-soluble to provide a conjugate acceptable for in vivo administration.
Other previously described polymeric reagents suffer from similar deficiencies.
Thus, there remains a need in the art to provide polymeric reagents that are useful for providing, among other things, conjugates with other substances. The present invention addresses this and other needs in the art by providing, inter alia, novel polymeric reagents comprising a ketone or a related functional group (e.g., a ketone hydrate, thione, monothiohydrate, dithiohydrate, hemiketal, monothiohemiketal, dithiohemiketal, ketal, or dithioketal).